title: Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2.
abstract: Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzyme's catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC(50) of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC(50) of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2.
authors.affiliation: Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
authors.forename: Ka To
authors.initials: KT
authors.lastname: Shum
authors.forename: Eric Lik Hang
authors.initials: EL
authors.lastname: Lui
authors.forename: Sybil Cheuk Ki
authors.initials: SC
authors.lastname: Wong
authors.forename: Pok
authors.initials: P
authors.lastname: Yeung
authors.forename: Laiju
authors.initials: L
authors.lastname: Sam
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
authors.forename: Julian A
authors.initials: JA
authors.lastname: Tanner
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D054856:G-Quadruplexes; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase